Inhibitory Peptide of Mitochondrial μ-Calpain Protects against Photoreceptor Degeneration in Rhodopsin Transgenic S334ter and P23H Rats

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.


Introduction
Retinitis pigmentosa (RP) is a hereditary retinal degeneration characterized by night blindness, photophobia, gradual loss of the peripheral visual field, color blindness, and eventual visual disturbance. These symptoms are caused by progressive rod photoreceptor degeneration in the early stage, followed by eventual cone photoreceptor degeneration. The disease prevalence is about 1/4,000-5,000, and the condition is common around the world. The hereditary characteristics are heterogeneous, and characterized by autosomal-dominant (ADRP), autosomal-recessive (ARRP) or X-linked inheritance patterns. Recent molecular genetic studies have also revealed that more than 100 different genes are involved in or cause RP (Ret-Net: http://www.sph.uth. tmc.edu/retnet/disease.htm).
Our previous studies demonstrated that calcium ions, calpain, and AIF are the main causes of photoreceptor cell death in RCS rats in the early stages of retinal degeneration [1,6,7,11]. First, Yamazaki et al found that a low-voltage-activated calcium channel blocker, nilvadipine, preserves retinal morphology and functions in RCS rats [11]. Those results suggested that intracellular concentrations of calcium ions are elevated, and calpains, as calciumdependent cysteine proteases, are activated in the photoreceptor. Second, we showed that mitochondrial calpain is activated and truncates AIF, followed by the release of truncated AIF (tAIF) from the mitochondria into the nucleus in the initial stage of retinal degeneration in RCS rats [6]. It is well known that after truncation of AIF by mitochondrial m-calpain [12][13][14][15][16][17][18][19][20], tAIF can translocate from the mitochondrial inner membrane to the nucleus, where it facilitates chromatin condensation and largescale DNA fragmentation [21,22]. We also found that intravitreal injection of the calpain inhibitors ALLN and PD150606 at the time of mitochondrial calpain activation transiently inhibited nuclear translocation of tAIF and photoreceptor apoptosis [6]. Inhibition of the mitochondrial m-calpain-AIF pathway would thus provide significant benefit in the treatment of RP.
Recently, we found that a specific peptide inhibitor of mitochondrial m-calpain, Tat-mCL (another name for HIV-Nm), transiently prevents retinal degeneration and attenuation of electroretinogram (ERG) response following intravitreal injection or eye-drop application in RCS rats [7]. The RCS rat carries a mutation in the Mertk gene expressed in the retinal pigment epithelium (RPE), and this mutation has been characterized in ARRP [23]. However, because the mutation is only one of many gene mutations causing RP, we still do not know whether the results from that previous study [7] can be generalized to other types of RP associated with defects genes other than the Mertk gene, or are instead specific to RP caused by mutations in the Mertk gene. To obtain clues for solving this question, we need to examine the effects of Tat-mCL on RP models other than the RCS rat. Because RP is genetically highly heterogeneous, molecular mechanisms that lead to photoreceptor apoptosis may also differ according to the causative genes. The present study, therefore, examined the protective effects of Tat-mCL against retinal degeneration using other RP models, namely Rho transgenic S334ter and P23H rats, as well-known models for ADRP [24][25][26].
Calpains and/or AIF play a significant role in the photoreceptor degeneration of both S334ter and P23H rats [3,4]. Shinde et al demonstrated that calpains are activated and AIF is released from the mitochondria to the cytosol in the initial stage of photoreceptor cell death in S334ter rats [4]. Furthermore, Kaur et al reported that the calpain-dependent pathway, but not the caspasedependent pathway, contributes to photoreceptor cell death in P23H rats [3].
Accordingly, the purpose of the present study was to determine whether the mitochondrial m-calpain inhibitory peptide, Tat-mCL, protects against retinal degeneration in both S334ter and P23H rats. Because degeneration progresses more rapidly in S334ter rats than in P23H rats, we examined the short-term protective effects of Tat-mCL against photoreceptor cell death and function in S334ter rats, and long-term protective effects in P23H rats.

Animals
All experimental procedures were designed to conform to the Association for Research in Vision and Ophthalmology (ARVO) Statement for Use of Animals in Ophthalmic Vision Research and were approved by the Committee for the Use of Live Animals (Permit Number: M10016) at the Hirosaki University. Sprague-Dawley (SD) rats were purchased from Clea Japan (Tokyo, Japan), and used as wild-type (wt) controls. Homozygous S334ter (line 4) and P23H (line 2) Rho transgenic rats were generously provided by Dr. Matthew M. LaVail (University of California), and were housed at the Hirosaki University Graduate School of Medicine Animal Care Service Facility under a 12-h light (50 lux illumination) and 12-h dark (,10 lux illumination) cycle. Care was taken not to cause photoreceptor light damage to rats.

Synthesis of m-calpain C2L Domain Peptides
We separately synthesized Tat-mCL (GRKKRRQRRRPPQ-PDALKSRTLR, 23 aa; molecular weight (MW), 2857.37 Da) and its scramble peptide (GRKKRRQRRRPPQ-ASLRLDRPTK, 23 aa; MW 2857.37 Da), as described in our previous study [7]. Each peptide was synthesized by the fluorenylmethyloxycarbonyl method using an automated peptide synthesizer (Shimazdu PSSM-8; Shimazdu, Kyoto, Japan). The resulting peptides were purified by reverse-phase HPLC using a C18 column (Jupiter 250 mm610 mm; Phenomenex, Torrance, CA). The molecular weight and purity of each peptide was confirmed by MALDI-TOF mass spectrometry with a Voyager RP-DE (Applied Biosystems, Foster City, CA). Purity of each synthesized peptide was .95% as estimated from the relative absorbance by HPLC.

Subcellular Fractionation of Rat Retinas
Subcellular fractionation of S334ter or P23H rat retinas was performed as described [6,27]. All experimental procedures were carried out at 4uC. Rats were sacrificed with inhalation of carbon dioxide. After enucleation, eyes were washed in ice-cold phosphate-buffered saline (PBS) (0.14 M NaCl and 10 mM phosphate buffer, pH 7.4) and dissected into halves. Retinas taken from both eyes of each rat were homogenized in 500 ml of homogenizing buffer (20 mM Tris-HCl, pH 7.5, containing 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 0.25 M sucrose and 5 mM 2mercaptoethanol) with a 2-ml-glass-teflon homogenizer. The homogenate was centrifuged at 6006g for 5 min to remove the nuclear fraction, and the supernatant was then centrifuged at 4,5006g for 10 min to obtain the mitochondrial fraction. The supernatant was then centrifuged at 20,0006g for 20 min to remove the lysosomal fraction. The supernatant was used as a cytosolic fraction. The mitochondrial fraction was suspended in 200 ml of suspending buffer (20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol), and sonicated to disrupt the mitochondrial inner and outer membrane.

Western Blot Analysis
Western blot analyses were performed to detect and determine the quantity of AIF, as previously described [28,29]. Mitochondrial or cytosolic proteins of S334ter or P23H rat retinas were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on 10% SDS-polyacrylamide gels (20-40 mg proteins per lane). Western blotting was performed after SDS-PAGE. Immunoreactive signals were developed with an enhanced chemiluminescence Western blotting detection kit (Amersham Biosciences, Buckinghamshire, UK) and quantified with a luminescent image analyzer (LAS-3000; Fujifilm Co., Tokyo, Japan).

Administration of Tat-mCL
We determined the short-and long-term effects of Tat-mCL using S334ter and P23H rats, respectively. For S334ter rats, we performed intravitreal injection of 20 mM Tat-mCL at postnatal (PN) 15 days or topical eye-drop application of 20 mM Tat-mCL twice a day on PN 13-17 days for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and PN 13-55 days for ERG. For P23H rats, we performed topical eyedrop application of 1 mM Tat-mCL twice a day on PN 14-39 days for immunohistochemical study, PN 14-49 days for TUNEL assay, and PN 14-89 days for ERG and light microscopic studies. Assuming a volume of rat vitreous of 30 ml, the final concentration of peptide in the vitreous was considered to be 1.25 mM immediately after intravitreal injection. However, we estimated that the concentration of peptide in the mitochondria of photoreceptor cells would be within the range of one-tenth to one-hundredth of 1.25 mM, i.e, 12.5-125 mM, because the peptides would be diluted by ocular circulation, degraded by various proteases, and/or blocked by the several barriers such as extracellular matrices. These concentrations of 12.5-125 mM would be considerably higher than IC 50 (197 nM) [7] and would exert no cytotoxic effects on the photoreceptors. In topical eyedrop application, we used 20 mM Tat-mCL for S334ter rats and 1 mM peptide for P23H rats, respectively. The concentration of the peptide was determined according to the severity of each RP model [30]. Because the P23H (line 2) has been demonstrated to be slowly progressive [30], we speculated that 1 mM Tat-mCL might be sufficient.
Rats were anesthetized by intramuscular injection of ketamine (80-125 mg/kg) and xylazine (9-12 mg/kg). After topical application of 0.02% oxybuprocaine hydrochloride to the cornea, each animal received an intravitreal injection of 2 ml of 20 mM Tat-mCL in PBS, 4 mM PD150606, or PBS (n = 6 per group). Solutions were injected using a Hamilton syringe with a 30-gauge needle through the area of the ciliary body (1 mm posterior to the corneal limbus) into the vitreous cavity. Special care was taken to avoid injury to the lens and the needle was penetrated vertically into the eyeball. Alternatively, topical eye-drop application was performed twice a day without anesthesia.

TUNEL Assay
To determine retinal photoreceptor cell death, cleavage of DNA was visualized in situ by TUNEL assay as previously described [7]. The eyes of Tat-mCL-treated RCS rats were enucleated and embedded in OCT compound (Sakura, Tokyo, Japan). Cryosections (5 mm thick) were cut superior or inferior to the plane of the equator containing the optic disc and the central portion of the eyeball, respectively. Numbers of TUNEL-positive cells were counted in 20 sections per eye in nine eyes from each group. After washing with PBS, TUNEL was performed using In Situ Apoptosis Detection Kits (Takara, Ohtsu, Japan) according to the instructions from the manufacturer. Sections were counterstained with 49-69-diamidino-2-phenylindole (DAPI) to stain nuclei. Immunofluorescent images were acquired by laser scanning confocal microscopy (FV1000-D; Olympus, Tokyo, Japan).

Immunohistochemistry
Immunohistochemistry to detect the localization of AIF was performed as previously described [6]. Enucleated eyes were fixed in 4% paraformaldehyde in PBS, pH 7.4, for 20 min at room temperature. Anterior segments were removed and posterior eyecups were placed in the same fixative overnight at 4uC. Eyecups were cryoprotected for 4 h in 10% then 20% sucrose in PBS, then frozen in OCT compound. Cryosections (5 mm thick) were made superior or inferior to the equator plane containing the optic disc and the central portion of the eyeball. Sections were rinsed in PBS, incubated with 0.3% hydrogen peroxide in methanol for 15 min, and blocked with 1% skim milk in PBS plus Tween (PBS-T) for 2 h at room temperature. Sections were incubated overnight at 4uC with the rabbit polyclonal anti-AIF antibody (ab1998, 1:1000; Abcam, Cambridge, UK) diluted in 1% skim milk in PBS-T. Sections were then washed with PBS-T and incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (P0448, 1:500; DAKO, Glostrup, Denmark) overnight at 4uC. Sections were washed with PBS-T and incubated with tetramethylrhodamine-labeled tyramide (PerkinElmer. Ins, Waltham, MA) for 10 min at room temperature. Sections were then washed with PBS-T and mounted with Vectashield (Vector, Burlingame, CA) with DAPI.

ERG
The methods for measurement of ERG response were described in our previous study [7]. The time points for ERG measurement were determined according to the time courses of photoreceptor degeneration in S334ter (line 4) and P23H (line 2) rats [30]. ERGs were recorded at PN 18,21,28,35,42,49 and 56 days for S334ter rats, and at PN 30, 70 and 90 days for P23H rats, respectively. Briefly, S334ter or P23H rats were moved to an electrically shielded room, anesthetized, and laid on their side on a heating pad (at 37uC). The head was fixed in place with surgical tape, and the rats were dark-adapted for 24 h. The pupils were dilated with 0.5 mg/ml tropicamide and 0.5 mg/ml phenylephrine hydrochloride eye-drops. ERGs were recorded with a contact electrode equipped with a suction apparatus to fit on the cornea (Kyoto Contact Lens, Kyoto, Japan). A grounding electrode was placed on the nose. Responses to a 200-ms duration white flash (3.5 610 2 lux) were recorded (Neuropack, model MES-3102; Nihon Kohden, Tokyo, Japan). The a-wave amplitude was determined as the baseline to the bottom of the a-wave. The bwave amplitude was determined as the bottom of the a-wave to the top of b-wave. ERG amplitudes are shown as mean 6 standard deviation.

Statistical Analysis
Student's t test or analysis of variance (ANOVA) was used to statistically compare results. Experiments were performed in triplicate to confirm reproducibility.

TUNEL Assay in S334ter Rat Retinas
To determine the time course of the photoreceptor degeneration in S334ter rat retina, we performed the TUNEL assay to detect the photoreceptor cell death (Figure 1). In the outer nuclear layer (ONL), the TUNEL-positive cells were detected from PN 13 days and reached the highest level at PN 15 and 18 days, while photoreceptors were still undergoing cell death at PN 21-30 days.

Intravitreal Injection of Tat-mCL Prevents Photoreceptor Cell Death in S334ter Rats
With intravitreal injection, we determined the protective effects of Tat-mCL against photoreceptor cell death in S334ter rats (

Topical Eye-drop Application of Tat-mCL Protects Photoreceptors in S334ter Rats
We applied eye-drops containing Tat-mCL to S334ter rats, and examined the protective effects against photoreceptor degeneration ( Figure 3). We placed eye-drops containing vehicle (PBS) or 20 mM Tat-mCL on the eyes of S334ter rats twice a day from PN 13 to 17 days. The eyes were enucleated on PN 18 days and the TUNEL-positive cells were detected. The number of TUNEL-

Protective Effects of Tat-mCL on Retinal Function in S334ter Rats
Considering the protection against photoreceptor cell death with Tat-mCL, we determined the effects of Tat-mCL on preservation of retinal function in S334ter rats, using ERGs ( Figure 4). We also compared the effects of intravitreal injection and eye-drop applications on ERG responses. In the group with intravitreal injection, rats received an intravitreal injection of 2 ml of 20 mM Tat-mCL at PN 15 days. In the group with eye-drop applications, we placed eye-drops containing vehicle (PBS) or 20 mM Tat-mCL on the eyes of S334ter rats twice a day from PN 13 to 56 days. Scotopic ERGs were recorded on PN 18, 21, 24, 28, 35, 42, 49 and 56 days. Although a flash ERG was performed on the dark-adapted eye, response was primarily from the rod system. Sufficiently bright flashes elicit ERGs containing an awave (initial negative deflection), followed by a b-wave (positive deflection). The a-wave is derived from photoreceptors, while the leading edge of the b-wave is mainly produced by the Müller cells. Tat-mCL-injected retinas showed significant preservation of awave at only PN 28 and 35 days ( Figure 4A). However, retinas with Tat-mCL eye-drops showed significant preservation at PN 28,35,42,49 and 56 days in a sustained manner. Although no significant protection of b-wave was observed in Tat-mCL-injected retinas, eye-drop application of Tat-mCL significantly and persistently preserved the b-wave at PN 35,42,49 and 56 days ( Figure 4B).

Topical Eye-drop Application of Tat-mCL Protects Photoreceptors in P23H Rats
Next, we examined the protective effects of Tat-mCL on another model of Rho mutants, P23H rats. Because the retinal degeneration in P23H rats progresses slowly, we examined the long-term protective effects of eye drop application of Tat-mCL against retinal degeneration in P23H rats (Figures 5-8). We used saline (PBS) or 0.1% hyaluronic acid (HA) as solvent for Tat-mCL. Eyedrops comprising PBS, 1 mM Tat-mCL in PBS, or 1 mM Tat-mCL in 0.1% HA were administered from PN 14 to 49 days. The eyes were enucleated at PN 30, 40 or 50 days, and retinal sections were stained with TUNEL. Treatment with Tat-mCL in PBS or in HA decreased TUNEL-positive cells in the ONL at PN 30, 40 and 50 days ( Figure 5A). Quantitative analysis of the number of TUNEL-positive cells showed that Tat-mCL significantly prevented photoreceptor cell death ( Figure 5B).

Topical Eye-drop Application of Tat-mCL Inhibits Nuclear Trasnlocation of AIF in Photoreceptors of P23H Rats
AIF is known to be truncated by mitochondrial m-calpain and translocate from the mitochondria to the nucleus, where it facilitates chromatin condensation and large-scale DNA fragmentation [12,13,15,21]. We have previously shown that mitochondrial calpains are activated and truncate AIF, followed by the release of truncated AIF from the mitochondria into the nucleus in the initial stage of retinal degeneration in RCS rats [6]. Shinde et al also demonstrated a 6.5-fold increase in protein levels of truncated AIF in the cytoplasmic fraction of S334ter (line 4) compared to SD rat retinas on PN 15 days [4]. However, whether AIF is involved in photoreceptor cell death in P23H rats remains to be clarified. We therefore investigated whether AIF released from mitochondria accumulated in photoreceptor nuclei of P23H rats. We found that AIF was translocated from the mitochondriarich inner segment to some photoreceptor nuclei in P23H rat retinas at PN 40 days ( Figure 6A). In addition, one of the AIFpositive nuclei was stained with TUNEL ( Figure 6A, white arrows). Western blot analyses could not detect the release of tAIF from mitochondria into cytosol (data not shown), presumably because of the small amounts. In wild-type SD rat retinas, we could not detected AIF translocation to photoreceptor nuclei [6]. Next, we determined whether eye-drop applications of Tat-mCL prevented AIF translocation to photoreceptor nuclei in P23H rats. Eye-drops containing PBS, 1 mM Tat-mCL in PBS, or 1 mM Tat- mCL in 0.1% HA were administered from PN 14 to 39 days. Eyes were enucleated at PN 40 days, and retinal sections were stained with AIF. We found that eye-drop application of Tat-mCL prevented the nuclear translocation of AIF in ONL ( Figure 6B).

Topical Eye-drop Application of Tat-mCL Prevents Thinning of the Photoreceptor Layer in P23H Rats
We determined the severity of thinning of the photoreceptor layer and the protective effects of eye-drop application of Tat-mCL in P23H rats. Eye-drops containing PBS, 1 mM Tat-mCL in saline, or 1 mM Tat-mCL in 0.1% HA were administrated from PN 14 to 89 days. Eyes were enucleated at PN 30, 70 or 90 days, and retinal sections were stained with hematoxylin and eosin. The results showed that thickness of the ONL gradually decreased from PN 30 to 90 days ( Figure 7A). However, treatment with Tat-mCL clearly prevented thinning of the ONL. Quantitative analysis of ONL thickness showed that Tat-mCL significantly prevented thinning of the ONL at PN 70 and 90 days ( Figure 7B).

Topical Eye-drop Application of Tat-mCL Protects Retinal Function in P23H Rats
We examined the effects of Tat-mCL on preservation of retinal function in P23H rats using ERG (Figure 8). We placed eye-drops containing vehicle (PBS) or 1 mM Tat-mCL on the eyes of P23H rats twice a day from PN 14 to 89 days. Scotopic ERGs were recorded at PN 30, 70 and 90 days. ERG responses were seen gradually attenuate from PN 30 to 90 days ( Figure 8A). However, treatments of Tat-mCL noticeably prevented attenuation of ERG response at PN 70 and 90 days. Quantitative analysis of the a-and b-wave of ERG response showed that Tat-mCL significantly prevented attenuation at PN 70 and 90 days (Figures 8B, C).

Discussion
The present study demonstrated that mitochondrial m-calpain inhibitory peptide, Tat-mCL, prevented photoreceptor cell death and delayed the progression of retinal degeneration in Rho transgenic S334ter and P23H rats. Although we had previously found that Tat-mCL protects against retinal degeneration in Mertk mutant RCS rat [7], the present results revealed that the peptide also exerted protective effects against degeneration in the most prevalent mutations in ADRP, RHO mutants.
Although ADRP is associated with mutations in at least 20 different genes, mutations in the Rho gene (RHO, OMIM 180380,   accession ID U49742) are the most prevalent, identified in 30-40% of all ADRP cases [24,25]. In addition, Rho mutations also show highly variable phenotypes depending on the location of the mutation [25]. The mutation of Rho N-terminus, the substitution of histidine for proline in the 23 rd amino acid (P23H), has been observed in about 12% of ADRP patients [24]. Although P23H shows a relatively mild clinical progression, C-terminal mutations such as S334ter generally exhibit a more severe clinical phenotype [25,26]. This is probably because the C-terminal domain is important for Rho sorting to rod outer segments, and for Rho phosphorylation and binding of arrestin [31,32]. In P23H rats, Rho appears to be mis-folded in the ER [33,34]. In S334ter rats, Rho is truncated at the C-terminus and lacks the last 15 amino acid residues and is thus mis-localized in the cytoplasm or cell membrane of photoreceptors through interference with post-Golgi trafficking [32,35,36]. In any case, as a result of the inhibition of both sorting and dysfunction of Rho, photoreceptor physiology is changed and cell death results [2,[36][37][38][39][40][41].
The two different mutants show roughly comparable mechanisms of cell death mediated by calpains, AIF, ER stress, PARP, or caspase-3 [3][4][5]. Among these causative factors, the present study inhibited the mitochondrial m-calpain and AIF pathway using the Tat-mCL, because several studies have demonstrated that the activation of calpains and translocation of AIF from mitochondria occurred in the initial stage of photoreceptor cell death in S334ter and P23H rats [3,4]. Our results show that intravitreal injection or eye-drop application of the Tat-mCL inhibited photoreceptor cell death in the early stages of degeneration in S334ter and P23H rats (Figures 2, 3 and 5). In P23H rats, the peptide also prevented nuclear translocation of AIF in the photoreceptor ( Figure 6B). These protections would have beneficial effects in delaying the progression of visual disturbance and thinning of the photoreceptor layer (Figures 4, 7 and 8). Our results suggest that retinal degeneration occurs via the mitochondrial m-calpain and AIFdependent pathway in not only RCS rats, but also Rho transgenic S334ter and P23H rats. In addition, inhibition of this pathway would delay retinal degeneration in RP resulting from Rho gene mutations. The present results also suggest that inhibition of photoreceptor cell death by the Tat-mCL may be mutationindependent.
In contrast, we could not completely prevent the attenuation of ERG responses and thinning of the photoreceptor layer. In addition to inhibition of the mitochondrial m-calpain and AIF pathway, we should consider ER stress, oxidative stress, and caspase activation induced in the middle to late stages of retinal degeneration in S334ter and P23H rats. In particular, we should also take the mis-folding and mis-sorting of Rho protein into account. Recent studies have revealed that ER stress response or unfolded protein response (UPR) is involved in retinal degeneration in mouse, rat, and Drosophila models of RP [4,5,[42][43][44][45].
In addition to RP, the inhibition of calpain activity seems likely to be beneficial for protection against the retinal ganglion cell (RGC) death seen in glaucoma. Recent studies have shown that calpains are activated and inhibition of calpain activity attenuates RGC death in rat models of glaucoma [46][47][48]. Maintenance of RGC mitochondria is also key to neuroprotection in glaucoma [49]. Although it remains to be elucidated whether RGC degeneration involves the mitochondrial m-calpain and AIFdependent pathway, the intracellular Ca 2+ elevation in RGC [50] could trigger the activation of mitochondrial calpains as well as cytosolic calpains, and activated mitochondrial calpains would contribute to RGC death via AIF truncation/activation in glaucoma. We thus believe that Tat-mCL has therapeutic potential for preventing RGC degeneration in glaucoma. Very recently, Das et al demonstrated that calpain inhibition could prevent inflammation, apoptosis, and axonal degeneration in a rat model of acute optic neuritis, experimental autoimmune encephalomyelitis [51]. The Tat-mCL may also offer widespread effects in treating optic neuritis.
In summary, the mitochondrial m-calpain and AIF pathway is involved in the early stage of retinal degeneration in Rho transgenic S334ter and P23H rats, and inhibition of this pathway using Tat-mCL leads to effective treatment of RP involving Rho mutation.